A Genetic Analysis of the Drosophila Closely Linked Interacting Genes Bulge, Argos and Soba

The Drosophila gene argos encodes a diffusible protein that acts as a negative regulator of cell fate decisions. To define interacting gene products, we performed a genetic analysis of argos, which suggests the presence of several partially redundant gene functions in its immediate vicinity at the chromosomal position 73A. Dose titration experiments have identified two of these loci. One of them corresponds to the gene bulge. Loss of function bulge alleles suppress the rough eye phenotype associated with overexpression of argos; conversely, amorphic argos mutations suppress the eye phenotype seen in flies bearing a single dominant bulge allele. Recombination mapping localized bulge 0.15 cM distal to argos. A second gene, suppressor of bulge and argos (soba), corresponds to the recently described lethal complementation group 73Aj. soba alleles suppress the eye phenotypes seen in flies expressing either the dominant bulge allele or the hs-argos construct. soba resides 120 kb proximal to argos. In addition, we have identified one allele of a new gene, clown, which like soba suppresses the eye phenotypes associated with hs-argos and bulge(Dominant). clown maps on chromosome 3 at the cytological position 68CD.     

Misexpression of argos, an inhibitor of EGFR signaling in oogenesis, leads to the production of bicephalic, ventralized, and lateralized Drosophila melanogaster eggs

Epidermal growth factor receptor (EGFR) signaling pathways are frequently involved in generating cell fate diversity in a number of organisms. During anterior-posterior and dorso-ventral polarity in the Drosophila egg chamber and eggshell, EGFR signaling leads to a number of determinative events in the follicle cell layer. A high level of Gurken signal leads to the expression of argos in dorsal midline cells. Lateral follicle cells, receiving a lower level of Gurken signal, can continue to express the Broad-Complex (BR-C) and differentiate into cells which produce chorionic appendages. Misexpression of argos in mid-oogenesis causes the midline cells to retain expression of BR-C, resulting in a single fused large appendage. Evidence that argos can directly repress Gurken-induced EGFR signaling is seen when premature expression of argos is induced earlier in oogenesis. It represses the Gurken signal at stage 5-6 of oogenesis which determines posterior follicle cells and occasionally leads to eggs with anteriors at both ends. We propose that the Gurken signal at stage 9 of oogenesis induces follicle cells to take on two fates, dorsal midline and lateral, each producing different parts of the eggshell and that argos is one of the key downstream genes required to select between these two fates.     

EGF signaling and ommatidial rotation in the Drosophila eye

The ommatidia of the Drosophila eye initiate development by stepwise recruitment of photoreceptors into symmetric ommatidial clusters. As they mature, the clusters become asymmetric, adopting opposite chirality on either side of the dorsoventral midline and rotating exactly 90 degrees (Figures 1A and 1B, ). The choice of chirality is governed by higher activity of the frizzled (fz) gene in one cell of the R3/R4 photoreceptor pair and by Notch-Delta (N-Dl) signaling. The 90 degrees rotation also requires activity of planar polarity genes such as fz as well as the roulette (rlt) locus. We now show that two regulators of EGF signaling, argos and sprouty (sty), and a gain-of-function Ras85D allele, interact genetically with fz in ommatidial polarity. Furthermore, we find that argos is required for ommatidial rotation, but not chirality, and that rlt is a novel allele of argos. We present evidence that there are two pathways by which EGF signaling affects ommatidial rotation. In the first, typified by the rlt phenotype, there is partial transformation of the "mystery cells" toward a neuronal fate. Although most of these mystery cells subsequently fail to develop as neurons, their partial transformation results in inappropriate subcellular localization of the Fz receptor, a likely cue for regulating ommatidial rotation. Secondly, reducing EGF signaling can specifically affect ommatidial rotation without showing transformation of the mystery cells or defects in polarity protein localization.     

Role of the EGFR/Ras/Raf pathway in specification of photoreceptor cells in the Drosophila retina

The Drosophila EGF receptor is required for differentiation of many cell types during eye development. We have used mosaic analysis with definitive null mutations to analyze the effects of complete absence of EGFR, Ras or Raf proteins during eye development. The Egfr, ras and raf genes are each found to be essential for recruitment of R1-R7 cells. In addition Egfr is autonomously required for MAP kinase activation. EGFR is not essential for R8 cell specification, either alone or redundantly with any other receptor that acts through Ras or Raf, or by activating MAP kinase. As with Egfr, loss of ras or raf perturbs the spacing and arrangement of R8 precursor cells. R8 cell spacing is not affected by loss of argos in posteriorly juxtaposed cells, which rules out a model in which EGFR acts through argos expression to position R8 specification in register between adjacent columns of ommatidia. The R8 spacing role of the EGFR was partially affected by simultaneous deletion of spitz and vein, two ligand genes, but the data suggest that EGFR activation independent of spitz and vein is also involved. The results prove that R8 photoreceptors are specified and positioned by distinct mechanisms from photoreceptors R1-R7.     

Mutations modulating the Argos-regulated signaling pathway in Drosophila eye development

Argos is a secreted protein that contains an EGF-like domain and acts as an inhibitor of Drosophila EGF receptor activation. To identify genes that function in the Argos-regulated signaling pathway, we performed a genetic screen for enhancers and suppressors of the eye phenotype caused by the overexpression of argos. As a result, new alleles of known genes encoding components of the EGF receptor pathway, such as Star, sprouty, bulge, and clown, were isolated. To study the role of clown in development, we examined the eye and wing phenotypes of the clown mutants in detail. In the eye discs of clown mutants, the pattern of neuronal differentiation was impaired, showing a phenotype similar to those caused by a gain-of-function EGF receptor mutation and overexpression of secreted Spitz, an activating ligand for the EGF receptor. There was also an increased number of pigment cells in the clown eyes. Epistatic analysis placed clown between argos and Ras1. In addition, we found that clown negatively regulated the development of wing veins. These results suggest that the clown gene product is important for the Argos-mediated inhibition of EGF receptor activation during the development of various tissues. In addition to the known genes, we identified six mutations of novel genes. Genetic characterization of these mutants suggested that they have distinct roles in cell differentiation and/or survival regulated by the EGF receptor pathway.     

The yeast TEM1 gene, which encodes a GTP-binding protein, is involved in termination of M phase.

LTE1 belongs to the CDC25 family that encodes a guanine nucleotide exchange factor for GTP-binding proteins of the ras family. Previously we have shown that LTE1 is essential for termination of M phase at low temperatures. We have identified TEM1 as a gene that, when present on a multicopy plasmid, suppresses the cold-sensitive phenotype of lte1. Sequence analysis of TEM1 and GTP-binding analysis of the gene product revealed that TEM1 encodes a novel low-molecular-weight GTP-binding protein. The defect of TEM1 was lethal, and the tem1-defective cells were arrested at telophase with high H1-kinase activity under restrictive conditions, indicating that TEM1 is required to exit from M phase. The defect of TEM1 was suppressed by a high dose of CDC15, which encodes a protein kinase homologous to mitogen-activated protein kinase kinase kinases. The genetic interaction among LTE1, TEM1, and CDC15 indicates that they cooperatively play an essential role for termination of M phase.     

Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate.

Pseudomonas putida PRS2000 is chemotactic to 4-hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-hydroxybenzoate at a high pH. The finding that 4-hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways.     

cDNA cloning of a novel cell adhesion protein expressed in human squamous carcinoma cells

A novel protein (SQM1 protein) present in human squamous epithelial cells has been found to be involved in cell adhesion in squamous epithelial cells, endothelial cells and extracellular matrix proteins. The corresponding cDNA that encodes a 135-residue polypeptide has been isolated. Sequence analysis indicates that the encoded polypeptide is distinct yet related to the beta subunit of integrins. A new sequence motif consisting of a heptadic repeat of positively-charged residues present in the polypeptide has been identified and is proposed to be important in protein-protein interaction. These results suggest that SQM1 protein, a new cell adhesion molecule expressed in squamous epithelial cells, may play an important role in tumor metastasis.     

The hierarchical relationship among the spitz/Egfr signaling genes in cell fate determination in the Drosophila ventral neuroectoderm

The spitz class and Egfr signaling (spi/Egfr) genes are required for the proper establishment of cell fate in the Drosophila ventral neuroectoderm. We investigated the role of the central nervous system (CNS) midline cells, and the hierarchical relationship among the spi/Egfr genes, in this process by analyzing the spatial and temporal expression of several of the genes in selected spi/Egfr mutants. Our analysis showed that expression of all the spi/Egfr genes is severely reduced in the single-minded (sim) mutant, and ectopically induced in en-Gal4/UAS-sim embryos. This result indicates that sim acts upstream of all the other spi/Egfr genes. The CNS midline cells regulate rhomboid (rho) expression in the ventral neuroectoderm and activate the EGFR signaling pathway. We also found that argos (aos) and orthodenticle (otd) act downstream of pointed (pnt), and that aos represses expression of otd in the lateral neuroectoderm to establish differential cell fates in the ventral neuroectoderm. Our findings suggest the following hierarchical relationship among the spi/Egfr genes: [see text].     

HuBMSC-MCP, a novel member of mitochondrial carrier superfamily, enhances dendritic cell endocytosis

A novel member of mitochondrial carrier superfamily has been identified from human bone marrow stromal cells (BMSC) and designated as human BMSC-derived mitochondrial carrier protein (HuBMSC-MCP). It encodes a 321 amino-acid protein with three tandem related domains of about 100 amino acids. Each domain contains two hydrophobic stretches, which are thought to span the membrane as alpha-helices. Distant relationship analysis indicates that the protein is highly conserved between species from Caenorhabditis elegans to human. HuBMSC-MCP gene is mapped to chromosome 11p11. HuBMSC-MCP mRNA expression is detectable in various human tissues and cell lines. By confocal imaging, HuBMSC-MCP is localized to mitochondria and also detected in the pseudopodial protrusion of human breast adenocarcinoma MCF-7 cells. When transfected into dendritic cells (DC), HuBMSC-MCP could enhance DCs endocytotic capacity. Thus, HuBMSC-MCP is a phylogenetically conserved and widely expressed mitochondrial carrier protein which perhaps associates with mitochondrial oxidative phosphorylation.     

Primary structure of human protein pS2

We have previously reported that pS2 mRNA expressed in cultured epithelial cells derived from a hormone-dependent breast carcinoma (MCF-7 cells) is also expressed in mucosa cells of normal human stomach. This mRNA encodes a putative 84 amino-acid-long protein, which is secreted by both cell types after elimination of a signal peptide. We report here the purification of the pS2 protein, its trypsin digestion and amino-acid sequencing. The MCF-7 cell-secreted protein is 60 amino-acid-long and its sequence is in complete agreement with that deduced from the mRNA sequence. The presence of an N-terminal glutamic acid indicates that the signal peptidase releases a 24 amino-acid-long signal peptide. Analysis of tryptic peptides derived from the secreted gastric pS2 protein indicates that the signal peptide and the sequence of the first 48 amino-acids are identical to those of secreted MCF-7 pS2 protein, although the N-terminal amino-acid of the gastric protein may be cyclized as a pyroglumatic acid.     

Metaphase arrest with centromere separation in polo mutants of Drosophila

The Drosophila gene polo encodes a conserved protein kinase known to be required to organize spindle poles and for cytokinesis. Here we report two strongly hypomorphic mutations of polo that arrest cells of the larval brain at a point in metaphase when the majority of sister kinetochores have separated by between 20-50% of the total spindle length in intact cells. In contrast, analysis of sister chromatid separation in squashed preparations of cells indicates that some 83% of sisters remain attached. This suggests the separation seen in intact cells requires the tension produced by a functional spindle. The point of arrest corresponds to the spindle integrity checkpoint; Bub1 protein and the 3F3/2 epitope are present on the separated kinetochores and the arrest is suppressed by a bub1 mutation. The mutant mitotic spindles are anastral and have assembled upon centrosomes that are associated with Centrosomin and the abnormal spindle protein (Asp), but neither with gamma-tubulin nor CP190. We discuss roles for Polo kinase in recruiting centrosomal proteins and in regulating progression through the metaphase-anaphase checkpoint.     

Absence of DNA ligase IV protein in XR-1 cells: evidence for stabilization by XRCC4

XR-1 is a CHO mutant cell line defective in double strand break repair and V(D)J recombination. These defects are due to a deletion of the XRCC4 gene which encodes a 38-kDa nuclear phosphoprotein. Recent studies have shown that XRCC4 interacts with and enhances the activity of DNA ligase IV in vitro. In this study we investigate the effect of the absence of XRCC4 on the level of DNA ligase IV in XR-1 cells. Western blot analysis indicates that levels of DNA ligase IV protein are almost undetectable in these cells, however, introduction of the XRCC4 cDNA into XR-1 resulted in a return to wild type levels of the protein. Furthermore, analysis of DNA ligase IV mRNA showed equivalent levels in both XR-1 and XRCC4 transfected XR-1 indicating that the altered level of DNA ligase IV is not due to a change in the expression of the gene. These data strongly suggest that an important function of XRCC4 is to stabilize the DNA ligase IV protein.     

Cloning and characterization of a cysteine proteinase from Saccharomyces cerevisiae.

We have isolated a gene from Saccharomyces cerevisiae that encodes a protein homologous to the mammalian cysteine proteinase bleomycin hydrolase. Sequence comparison between the yeast and rabbit proteins indicates an amino acid identity of 41.5% over 277 residues and a similarity of 78.3% when conservative substitutions are included. The apparent mass of the yeast protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 47 kDa, although sequence analysis indicates two potential initiator methionines that suggest calculated masses of either 51 or 55 kDa. The protein is nonessential in yeast as haploid mutants disrupted at several positions along the open reading frame remain viable. Furthermore, these mutants do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients, osmotic strength, or exogenous bleomycin. However, the purified protein does exhibit marked hydrolytic activity toward the substrate arginine 4-methyl-7-coumarylamide (Km = 12.8 microM, Vmax = 2.56 mumol mg-1 h-1), and yeast cells engineered to express this protein at higher levels maintain increased resistance to bleomycin compared to wild-type cells. Because this protein represents the first example of a cysteine proteinase identified in yeast, we have named it Ycp1 (yeast cysteine proteinase).     

A DNA Construct That Encodes the Rabies Virus Consensus Glycoprotein with a Proteasome Degradation Signal Induces Antibody Production with IgG2A Subtype Predominance

The possibility of enhancing the immunogenicity of the rabies virus glycoprotein antigen encoded by a DNA vaccine has been investigated. Ubiquitin-like protein FAT10 has been attached to the N-terminus of the glycoprotein to target it to the proteasome and stimulate its presentation by MHC class I. Two forms of the protein, chimeric and original, have been detected in cells transfected with the DNA construct encoding the chimeric protein. The presence of the glycoprotein on the cell surface has been detected by immunostaining of transfected cells. The production of IgG and IgG2a antibodies has been more efficiently induced in mice immunized with the plasmid that encodes the chimeric protein than in those immunized with the plasmid that encodes unmodified glycoprotein. Moreover, the level of IgG2a antibodies exceeded the level of IgG1 antibodies, which indicates a preferential increase in the Th1 component of the immune response. The proposed DNA construct that encodes a modified glycoprotein with a proteasome degradation signal may be a promising DNA vaccine immunogen for post-exposure prophylaxis of rabies.